Identification of Genes Required for Swarming Motility in Bacillus subtilis Using Transposon Mutagenesis and High-Throughput Sequencing (TnSeq)

Bacillus subtilis exhibits swarming motility, a flagellar-mediated form of surface motility. Here, we use transposon mutagenesis and sequencing (TnSeq) to perform a high-throughput screen for candidate genes required for swarming. The TnSeq approach identified all of the known genes required for flagellar biosynthesis and nearly all of the previously reported regulators that promote swarming. Moreover, we identified an additional 36 genes that improve swarming and validated them individually. Among these, two mutants with severe defects were recovered, including fliT, required for flagellar biosynthesis, and a gene of unknown function, yolB, whose defect could not be attributed to a lack of flagella. In addition to discovering additional genes required for B. subtilis swarming, our work validates TnSeq as a powerful approach for comprehensively identifying genes important for nonessential processes such as colony expansion on plates. IMPORTANCE In TnSeq, transposons are randomly inserted throughout the chromosome at a population level, but insertions that disrupt genes of essential function cause strains that carry them to fall out of the population and appear underrepresented at the sequence level. Here, we apply TnSeq to the nonessential phenotype of motility in B. subtilis and spatially select for cells proficient in swarming. We find that insertions in nearly all genes previously identified as required for swarming are underrepresented in TnSeq analysis, and we identify 36 additional genes that enhance swarming. We demonstrate that TnSeq is a powerful tool for the genetic analysis of motility and likely other nonlethal screens for which enrichment is strong.